蛇毒神经生长因子对神经损伤后脊髓前角运动神经元超微结构的保护作用

目的：研究周围神经损伤后神经生长因子对运动神经元的保护作用。方法：采用定位、定量、定时的方法钳夹大耳白免右坐骨神经,于损伤处注射蛇毒 ＮＧＦ ４００ Ｂｕ·ｋｇ－１·ｄ－１,神经损伤术后 １,３,７ｄ和 ２,３,４,６,８周动态观察脊髓腰段伤侧第 Ⅸ板层外侧群的大型运动神经元的超微结构变化,并对线粒体体密度变化进行定量分析。结果：神经损伤术后对照组超微结构的改变比实验组明显加重,实验组超微结构的恢复比对照组快。结论;ＮＧＦ对神经损伤后脊髓前角运动神经元超微结构的恢复有促进作用,从而对运动神经元可起一定的保护和促进恢复作用。     

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.     

Re-expression of Nerve Growth Factor Receptor after Axonal Injury Recapitulates a Developmental Event in Motor Neurons: Differential Regulation when Regeneration is Allowed or Prevented

Motor neurons in the brainstem and spinal cord transiently express nerve growth factor receptors (NGFr) during development, but not in normal adult animals. In this study, NGFr was immunohistochemically identified in hypoglossal motor neurons after different types of peripheral axonal injury in adult rats. NGFr is re-expressed in motor neurons 7 days after a nerve crush injury, and has disappeared again by 28 days. These times correspond, respectively, to the active phase of regeneration, and a time by which regeneration has largely been completed, as determined by electrical activation of tongue muscle twitch. In contrast, 7 days after nerve transection and ligation of the proximal stump to prevent regeneration, there is no re-expression of NGFr, but 28 days after such treatment NGFr is present in a few neurons. By this time, neuroma formation has begun proximal to the end of the cut and ligated nerve. Together, these findings suggest that motor neurons transiently re-express NGFr during regeneration and not in response to axonal transection per se. The signal triggering re-expression thus seems more likely to be the introduction of a message from the site of injury, rather than the loss of a target-derived message. Although the function of NGFr in developing and regenerating motor neurons is not known, its expression appears to be associated with periods of axonal growth and maturation.     

Targeted Delivery of Nerve Growth Factor via Fibronectin Conduits Assists Nerve Regeneration in Control and Diabetic Rats

This study examined the influence of fibronectin conduits joining two halves of a sectioned sciatic nerve, with and without preimpregnation with nerve growth factor, on regeneration in rats with streptozotocin-induced diabetes mellitus. Regeneration, measured morphometrically in fibres containing immunoreactivity to calcitonin gene-related peptide (CGRP) and GAP-43, was significantly less ( P < 0.0001 for all comparisons) in diabetic rats with fibronectin mat grafts without nerve growth factor compared to similarly treated controls. Regeneration distances in diabetic rats were reduced to 43% (CGRP-reactive fibres) and 44% (GAP-43-reactive fibres) of controls, and the total amounts of immunoreactivities in the conduits were also reduced, though by lesser amounts (55 and 61% of controls respectively). Impregnation of the conduits with nerve growth factor before implantation increased the distance and amounts of regenerating immunoreactivity in both control and diabetic rats for both CGRP and GAP-43, such that these regeneration parameters were similar in nerve growth factor-treated diabetic rats to those in control rats implanted with untreated fibronectin mat conduits. These findings implicate impaired neurotrophic support in the defective regeneration characteristic of diabetic neuropathy.     

神经生长因子对神经干细胞发育的调节

神经生长因子作为神经营养因子家族的成员之一，在神经系统中分布广泛。其除具备营养神经元、保护和修复受损神经等生物学效应外，对神经干细胞的增殖、存活、迁移和分化等过程也具有一定的影响。     

神经生长因子对大鼠前脑缺血再灌注后细胞凋亡及FaS蛋白表达的影响

目的：研究神经生长因子（NGF）对大鼠前脑缺血再灌注后海马CA1区Fas蛋白表达和细胞凋亡的影响。方法：夹闭大鼠双侧颈总动脉造成前脑缺血，30min后松夹再灌注，NGF组和生理盐水组于再灌注开始时分别肌肉注射NGF30μg·mL^-1和生理盐水0．1mL，应用免疫组化法和TUNEL法检测各组大鼠海马CA1区Fas蛋白表达和细胞凋亡。结果：再灌注后6和24h，NGF组Fas蛋白平均吸光度值小于生理盐水组（P〈0．001和P〈0．05）。再灌注后48h两组比较差异无统计学意义（P〉0．5）。再灌注后6、24和48h，NGF组TUNEL阳性细胞率均低于生理盐水组，差异有统计学意义（P〈0．005）。结论：NGF可以减少大鼠脑缺血再灌注后海马CA1区Fas蛋白表达，抑制细胞凋亡，从而发挥其神经保护作用。     

Effect of Recombinant Human Nerve Growth Factor on Cisplatin Neurotoxicity in Rats

In this study we evaluated the effect of recombinant human nerve growth factor (rhNGF) on cisplatin (CDDP)-induced sensory neuronopathy in an experimental paradigm in the rat. Young adult female Wistar rats were treated with CDDP (2 mg/kg ip twice weekly for nine times) alone or in combination with rhNGF (1 mg/kg sc on alternate days). The effect of CDDP +/- NGF treatment was evaluated with behavioral (tail-flick test) and neurophysiological (nerve conduction velocity in the tail) methods immediately after treatment and after a follow-up period of 6 weeks. Pathological and morphometrical examinations of the dorsal root ganglia (DRG) and sciatic and saphenous nerves were also performed. rhNGF treatment induced a significant reduction in the CDDP-induced decrease in nerve conduction velocity (P < 0.05), and this was associated with a significant protection against the decrease in somatic (P < 0.05), nuclear (P < 0.05), and nucleolar size (P < 0.01) caused by CDDP treatment. However, for each of the parameters examined the neuroprotection obtained with rhNGF treatment was not complete. At the follow-up examination no differences between the three groups were observed in tail-flick test and nerve conduction velocity. We conclude that rhNGF, administered according to the schedule used in this experiment, exerts a biologically significant neuroprotective effect against CDDP peripheral neurotoxicity.     

Nerve Growth Factor, Central Nervous System Apoptosis, and Adrenocortical Activity in Aged Fischer-344/Brown Norway F1 Hybrid Rats

During aging there is a progressive loss of neuronal function in the basal forebrain that results in cognitive impairment and cholinergic deficits. While altered neurotrophin (NT)-mediated signal transduction may account for some age-associated deficits, there are differences in the extent of NT responsiveness among different laboratory rat strains. Here we measured nerve growth factor (NGF) protein levels and fragmented DNA in the CNS, and basal and NGF-stimulated activity levels of the hypothalamus-pituitary-adrenocortical axis (HPAA) in 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. Our results show that while there is no age-associated differences in NGF protein levels, in aged Fischer-344/Brown Norway rats, there are increases in levels of immunoreactive fragmented DNA in the CNS and in adrenocortical responses to the peripheral administration of NGF. These data contribute to the characterization of the Fischer-344/Brown Norway F1 hybrid rat and provide baseline values useful for future studies on aged CNS.     

Nerve Growth Factor Treatment Increases Stimulus-evoked Release of Sensory Neuropeptides in the Rat Spinal Cord

In the adult rat, nerve growth factor (NGF) upregulates the nociceptive peptide substance P (SP) and calcitonin gene-related peptide (CGRP) in neuronal cell bodies of C fibres but the effects of NGF on release of these neuropeptides from the central afferent terminals have not been reported. Thus, this study compared rats treated with six doses of NGF over 2 weeks with controls and measured the release of the neuropeptides, SP and CGRP from the dorsum of the lumbar spinal cord in vitro. NGF (1.0 mg/kg s.c.) greatly increased basal and stimulus-evoked SP and CGRP release; at 0.1 mg/kg, NGF did not affect SP release but significantly increased CGRP basal outflow and evoked release. In a different set of experiments, topical application of NGF (100 ng/ml) to cords from naive rats did not increase stimulus-evoked release of SP or CGRP. These findings show marked stimulation by NGF treatment in vivo of release of sensory neuropeptides during stimulation of dorsal roots, albeit at relatively large doses of the neurotrophin.     

肝细胞生长因子对大鼠坐骨神经损伤保护作用的实验研究

目的探讨肝细胞生长因子(HGF)对周围神经损伤的保护作用。方法以Wistar大鼠坐骨神经捻挫为动物模型,用HGF局部给药,设立对照组。于周围神经损伤后不同时期进行感觉神经传导速度(SCV)、病理形态计量学图像分析、坐骨神经功能指数(SFI)及电镜下超微结构研究。结果HGF组SCV、SFI、有髓纤维密度高于对照组(P<001)。电镜下HGF组髓鞘的厚度、轴突直径高于对照组。结论HGF能促进大鼠坐骨神经损伤后的有髓纤维再生,并促进感觉、运动神经纤维恢复,是一种有效的治疗周围神经损伤的药物。     

NGF Depletion Reduces Ipsilateral and Contralateral Trigeminal Satellite Cell Reactions after Inferior Alveolar Nerve Injury in Adult Rats

Following peripheral nerve injury, neuronal cell functions in sensory ganglia shift from normal maintenance and neurotransmission toward survival and regeneration. A rapid modulation of glial cell activity, which is related to changes in neuronal-support cell interaction, also occurs after nerve injury. Nerve growth factor (NGF) is required for the survival and maintenance of specific populations of sensory and sympathetic neurons, and changes in neuronal gene expression after axonal injury are due in part to a loss of NGF retrograde transport from the periphery to the cell body. A similar role for NGF in modulating support cell responses to peripheral nerve injury, however, has not been demonstrated. Using an autoimmune model, we assessed the effects of NGF depletion in adult rats on the injury-induced expression of glial fibrillary acid protein immunoreactivity (GFAP-IR) in the ipsilateral and contralateral trigeminal ganglia (TG). Unilateral inferior alveolar nerve crush resulted in abilateral,NGF-dependent trigeminal satellite cell response. In control rats there was a widespread induction of GFAP-IR in the ipsilateral as well as the contralateral TG. In contrast, GFAP-IR was reduced to the mandibular division of the ipsilateral TG in NGF-depleted rats, and the contralateral up-regulation of GFAP-IR was entirely abolished. Bilateral sympathectomy failed to mimic the effects of autoimmunization. Our results provide evidence that NGF depletion inhibits injury-induced satellite cell responses, independent of its effects on sympathetic nerve function.     

包涵体中重组人神经生长因子的纯化,复性及鉴定

目的利用简单的纯化工艺，获得具有生物活性的重组人神经生长因子（rhNGF）。方法利用PET－11d－NGF／BL21工程菌表达rhNGF，通过改变培养基成份优化发酵条件。建立了一种在提取包涵体后经一步强阳离子柱层析的简单、快速、重复性好的rhNGF纯化方法。纯化产物用鸡胚背根神经节伸长法测定其生物学活性。结果优化发酵条件后，表达量提高至约占总菌体的10．9％。经一步层析，重组蛋白的纯度可达到80％以上。1BU（生物活性单位）约为0．5μg／mL。结论包涵体复性表明，包涵体的复性与复性液中氧化型与还原型含疏基化合物的比例密切相关。抑制杂菌生长，可明显提高rhNGF的表达量。纯化的rhNGF为分析NGF结构与功能及探讨其临床应用提供了材料。     

神经生长因子对大鼠脑缺血再灌注海马神经细胞损伤的影响

目的 :观察神经生长因子 (NGF)对脑缺血再灌注后海马CA1区神经细胞损伤的影响。方法 :采用大鼠脑缺血再灌注模型 ,在光镜和透射电镜下观察NGF治疗组和缺血再灌组动物脑缺血 3 0min再灌注 2 4h和 72h时海马组织学及超微结构的改变。结果 :在缺血 3 0min再灌注 2 4h和 72h时 ,NGF治疗组海马CA1区神经细胞的结构损伤与缺血再灌组比较显著减轻 ;NGF可以减轻海马迟发性神经细胞死亡 (DND)性损伤。结论 :NGF对缺血再灌注所致的神经细胞损伤可能具有一定的保护作用     

Human Recombinant Nerve Growth Factor Replaces Deficient Neurotrophic Support in the Diabetic Rat

Manipulation of neurotrophic support is a developing strategy for new therapy aimed at neurodegenerative diseases. This study demonstrates reduced content and retrograde transport of endogenous nerve growth factor (NGF) in sciatic nerve of diabetic rats. There were also reductions in the diabetic rats in NGF protein and mRNA in skin and muscle of the hindlimb. These deficits correlated with reductions in substance P and calcitonin gene-related peptide–both products of NGF-influenced genes in primary afferents. These manifestations of deficient neurotrophic support were corrected by intensive insulin treatment and surmounted by administration of exogenous human recombinant NGF in a dose-related manner. Impaired neurotrophic support may, therefore, participate in the pathogenesis of diabetic and other peripheral neuropathies.     

腺病毒载体介导的RNAi对胶质瘤U251细胞肝细胞生长因子受体表达的影响

目的探讨腺病毒载体介导的RNA干扰技术对胶质瘤细胞肝细胞生长因子（HGF）受体（c—Met）的表达和细胞凋亡的影响。方法用PCR法获得人U6启动子及带有c—Met反向互补靶序列的片段HU6shmet：利用腺病毒载体将其传递至U251细胞；分别采用RT-PCR和Western blot方法检测U251细胞的c—Met mRNA和蛋白的表达．Annexin—V—PI双染法流式细胞术检测细胞的凋亡状况。结果获得了带有人U6启动子及c—Met反向互补靶序列的重组腺病毒载体rAdUshmet1和rAdUshmet2。转导了rAdUshmet1和rAdUshmet2的U251细胞的c—Met mRNA和蛋白相对表达量较未转导腺病毒及转导了rAdGFP和rAdU-sicon的U251细胞均有明显下降（P〈0．01），rAdUshmet1和rAdUshmet2转导的U251细胞凋亡率分别为（13．5±4．21％和（28．2±5．6）％，明显高于未转导腺病毒的及rAdGFP和rAdUsicon转导的U251细胞凋亡率（P〈0．05）。结论腺病毒载体rAdUshmet通过抑制HGF受体c—Met表达，能在一定程度上阻断HGF—c—Met信号通路，有可能成为对胶质瘤进行基因治疗的有效载体。     

神经生长因子对红藻氨酸诱导癫痫发作大鼠海马神经细胞凋亡的影响

目的　探讨外源性神经生长因子 (NGF)对癫痫发作所致海马神经细胞凋亡有无抑制作用。方法　采用红藻氨酸 (KA)诱导癫痫大鼠模型 ,以原位末端标记法 (TUNEL)标记 DNA片段 ,检测凋亡细胞在大鼠海马CA1区的动态变化及 NGF对其的影响。结果　海马 CA1区凋亡细胞在实验 2 4 h出现 ,4 8h明显增多 ,于 72h达高峰 ,7d时最少。给予 NGF脑室内注射后 ,在各相应时间点凋亡细胞数均明显减少 (P<0 .0 1)。结论　外源性 NGF能抑制癫痫发作所致的海马神经细胞凋亡 ,NGF对癫痫脑损伤有保护作用。     

The induction of neurotrophin and trk receptor mRNA expression during early avian embryogenesis

Nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, designated neurotrophins, are a family of neurotrophic factors, having important functions in the survival of embryonic and adult neuronal subpopulations. Through the trk family of receptors, these neurotrophins utilize phosphotyrosine-mediated signal transduction. We have used RT-PCR to detect the expression of mRNA for the above neurotrophins and their respective receptors, namely trkA, trkB and trkC in embryonic stages 1–8 of chicken development. While trkA and trkC mRNAs were expressed from stage 1 onwards, NGF and NT-3 mRNAs were expressed only at stages 3 and 5, respectively. In contrast, BDNF mRNA was expressed at stage 1, being the only neurotrophin expressed prior to expression of its respective receptor trkB. However, the latter was not expressed until stage 8. These results indicate an earlier expression of some but not all trk proto-oncogenes, suggesting that the two different receptor mRNAs expressed i.e. trkA and trkC in conjunction with BDNF, at stage 1, may act in aspects of very early embryonic development, such as gastrulation. Thereafter, mRNAs for trkB, NGF and NT-3 are expressed reflecting their later action in early embryonic development.     

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor,Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.     

Similarities and Discrepancies in the Signaling Pathway for Nerve Growth Factor in an Insulin Producing Cell Line and a Neural Crest-Derived Cell Line

Like neuronal cells, insulin producing cells (beta cells) possess nerve growth factor (NGF) binding sites and express mRNA coding for the low- and high-affinity NGF receptors, p75^NGFR and Trk-A respectively. Although the role of NGF on neuronal cells is well documented, its function on beta cells is still unknown. As a first step towards the elucidation of the role of NGF on beta cells, we have characterized both types of NGF receptors on INS-1 cells, a beta cell line derived from a rat insulinorna and studied some early post-receptor events by comparing the signaling pathway of NGF in those cells and in PC12 cells, a well characterized NGF-responsive cell line. By polymerase chain reaction, immunocytochemistry, cross-linking and Western blot analysis, we clearly demonstrated that Trk-A and p75^NGFR, the two NGF receptors expressed in INS-1 cells and PC12 cells are similar. Moreover, upon NGF treatment, Trk-A is phosphorylated on tyrosine residues in both cell types in the same dose- and time-dependent manner. These data clearly demonstrate that the first step of NGF signal transduction is similar in PC12 and INS-1 cells. Although early responsive genes like NGFI-A and c-fos are induced in both cell types upon NGF treatment, the induction of c-jun expression is restricted to PC12 cells. Furthermore, the expression of late responsive genes, such as vgf and transin, which are induced by NGF in PC12 cells, are not induced in INS-1 cells. In conclusion, although the initial steps of NGF signal transduction are similar in PC12 and INS-1 cells, some of the later differ. These dissimilarities could suggest that NGF plays different roles in neuronal and pancreatic beta cells.